Isolation of genomic DNA from lymphocytes/mammalian cell lines

(by phenol chloroform isoamyl alcohol (PCI) method)

Aim:
To isolate genomic DNA from lymphocytes/human liver cell line by phenol chloroform
isoamyl alcohol method.

Introduction:
Genomic DNA isolation is the fundamental requirement for genome characterization for
gene mapping, gene sequencing and for identification and isolation of genes for genetic
engineering and forensics.
In humans and animals, the genomic DNA can be obtained from certain bodily fluids
such as blood, and tissues or from cultured cell lines. The extraction of DNA can be
carried out using calcium chloride density gradient, anion exchange method, and crude
lysate and salting out method.
Principle:
Extraction of DNA consists of the following steps:
1) Preparation of a cell extract – Extraction buffer (EDTA and SDS) used to disrupt
the cell membrane. After lysis, the cell debris can be centrifuged to obtain cell as
clear supernatant.
2) Purification of DNA from cell extract – To remove the proteins/lipids present in
the cell extract by addition of buffer saturated phenol and mixture of chloroform
and isoamyl alcohol.
3) Concentration of DNA samples – Ethanol precipitation is the method used to
precipitate nucleic acids in the presence of salt (10M ammonium acetate).

Add SDS, EDTA,

Proteinase K,
RNAse

DNA
Requirements:

1. Human lymphocytes (or human liver cell line-HepG2),

2. Trypsin
3. Phosphate buffer saline (PBS),
4. DNA extraction buffer (TEN buffer): (50mM Tris-HCl, 10mM NaCl, 10mM
EDTA pH 7.5) [can also contain 10% (w/v) Sodium dodecyl sulphate (SDS)]
5. Proteinase K (20mg/ml),
6. RNAse A (20mg/ml),
7. Buffer saturated phenol (pH 8.0)
8. Chloroform:isoamyl alcohol (24:1)
9. Absolute ethanol
10. 10M ammonium acetate pH 5.2
11. 70% ethanol
12. MilliQ water or TE Buffer (Add 10ml of 1M Tris-Cl and 2ml of 0.5M EDTA,
make up to 100ml)

Protocol:

1) From T25 flask 0.5 ml of lymphocytes culture was transferred to microfuge tube
and centrifuged for 5 minutes at 5000 rpm. (Cultured human liver cells (HepG2)
were scraped from the T25 tissue culture flask and transferred to clean tube and
centrifuged for 5 minutes at 5000 rpm)
2) Supernatants were discarded and 1 ml of PBS was added to the pellet and
resuspended, centrifuged again as before. Discard the supernatant.
3) The cells were washed again with 1 ml of PBS (optional).
4) 0.5ml of DNA extraction buffer was added to the cell pellet and gently mixed to
resuspend the cells.
5) To this 5 μl of proteinase K and 1 μl of RNAse were added and incubated at 37°C
overnight.
6) Add equal volume of buffer saturated phenol. The tubes were then inverted for 15
minutes to ensure thorough mixing.
7) The tubes were centrifuged at 12000rpm for 15 minutes at 4°C.
8) The upper layer was carefully collected and transferred to a fresh tube. A wide-
bore pipette was used for the transfer as the contents will be sticky. (This will
avoid shearing of DNA.)
9) To the tubes, equal volume (500μl) of chloroform and isoamyl alcohol (24:1) was
added and continuously mixed at room temperature (using Roto-spin) for
15minutes.
10) The tubes were centrifuged at 12000rpm for 15 minutes at 4°C and the upper
aqueous layer was collected transfer to fresh tube.
11) To the tubes, 1/5th the volume of 10M ammonium acetate and double the volumes
of 100% chilled ethanol were added. The contents were mixed gently and kept at -
20°C overnight or -80°C for 1-2 hours.
 12) The tubes were centrifuged at 12000 rpm for 10-15 minutes at 4°C and the
supernatant was discarded.
13) 0,5 ml of 70% ethanol was added to wash the pellet (to remove excess salts) by
gently dissolving the pellet.
14) The tubes were centrifuged 12000 rpm for 15 minutes and the supernatant
discarded.
15) The tubes were dried by keeping their caps open on the bench top (inverted),
ensuring not to over dry the pellet, as it dissolution of DNA becomes difficult.
16) The DNA was dissolved in appropriate amount (25-50µl) of TE buffer/MilliQ
water and kept on the bench till it completely dissolved.
17) The tubes were stored at -20°C without vortexing until further analysis.

Basics of the DNA extraction procedure

In this experiment, DNA is isolated from the human lymphocytes. Cells were lysed using
extraction buffer (TEN/TNE), which contains Tris, EDTA, NaCl and additionally, the
buffer can also contain detergents such as SDS. The pH of buffer is maintained at 8 by
Tris, which is a buffering agent and also permeabilizes the cell membrane. EDTA,
chelating agent, stops interference of cellular ions, especially of those required for the
action of DNases enzyme thus blocking their activity. NaCl prevents the DNA from
denaturation. Na+ ions bind to the negatively charged phosphate groups in the DNA
backbone which causes them to attract to one another. This change in polarity will make
the DNA insoluble in water. Sodium chloride helps to remove proteins that are bound to
the DNA. It also helps to keep the proteins dissolved in the aqueous layer. SDS, anionic
detergent that helps in the denaturation of cell membrane by removing the lipids and
denatures all proteins having the potential of affecting DNA during extraction.

Alkaline pH of buffer causes cell lysis. Further addition of proteinase K degrades the
proteins present in the cell. The cells are treated with buffer saturated phenol and
chloroform-isoamyl alcohol (24:1) solutions for separation of DNA from lipid and
proteins present in the cells. In vivo, proteins are surrounded by water, a polar solvent,
with the hydrophobic amino acids buried inside and away from contact with water. When
treated with phenol, a non-polar organic solvent, a switch in polarity results in protein
denaturation.
Nucleic acids are more soluble in polar solvents; addition of chloroform results in
physical separation of DNA from proteins (Chloroform, in which nonpolar proteins and
lipids get dissolved to promote the partitioning of lipids and cellular debris into the
organic phase, leaving isolated DNA protected in the aqueous phase).
Differences in the densities of phenol and chloroform to that of water, results in two
separate layers (organic and aqueous) after centrifugation so the DNA containing
aqueous layer can be separated out easily.
Isoamyl alcohol is an anti-foaming agent which further degrades proteins, fats and
dissolves them and also aids in clear phase separation.
The DNA is precipitated with chilled absolute ethanol (100% ethanol), which in the
presence of ammonium acetate and forms an insoluble complex with nucleic acids.
Precipitated DNA is pelleted and dissolved in TE buffer or MilliQ water and run on an
agarose gel to confirm successful DNA extraction. The genomic DNA obtained is stored
at 4°C or -20°C.
“Spooling" At the last step of DNA extraction, purified DNA precipitates out after the
addition of chilled ethanol. This DNA appears as collection of fine threads in the
suspension which can be spool over the glass rod, this method is called as DNA spooling.
It is well known that ethanol has a lower dielectric constant than water, making it to
promote ionic bond (phosphate groups with any positive ions) formations causing the
DNA to precipitate.
Chilled ethanol is widely used to reduce the temperature of precipitation for good quality
of DNA. Chilled ethanol is also used to reduce the temperature to prevent the activity of
DNAse.
By adding salt, we help neutralize the DNA charge and make the molecule less
hydrophilic, meaning it becomes less soluble in water.
70% ethanol is added to remove excess salts, isolated DNA is stable at 4ºC upto one year
and more than 10years in -20 ºC

Observation: Genomic DNA will be subjected electrophoresis using 0.8% agarose gel
containing ethidium bromide and observed under UV light